Association of 12 serum biochemical markers of angiogenesis, tumour invasion and bone turnover with bone metastases from breast cancer: a crossectional and longitudinal evaluation.

Complex biological pathways including angiogenesis, invasion, osteoclastic activation and bone matrix degradation are involved in the formation of bone metastasis (BM). The aim of our study was to investigate the cross-sectional and longitudinal associations of a panel of 12 serum biochemical markers reflecting biological pathways underlying BM development. In a cross-sectional study, we investigated 29 patients with primary breast carcinoma without BM (BC/BM-), 28 patients with breast carcinoma and BM (BC/BM+) and 15 healthy women. In longitudinal analyses, we investigated 34 patients for whom serum was obtained a two different time points: at the time of primary BC diagnosis and after a median time of 3 years. During this follow-up, 15 patients developed BM, whereas the other 19 remained free of BM. In patients who developed BM, the second samples were obtained before BM was documented by bone scan. The cross-sectional analyses have shown all biochemical markers to be significantly elevated in patients with BM, when compared to the patients without BM and healthy controls, except TGFbeta1 that was significantly decreased. Multivariable analyses showed that only the bone resorption markers TRACP 5b, CTX and ICTP, and the marker of angiogenesis VEGF were independently associated with BM. Those markers correctly distinguished 85% of BC patients with or without BM from normal individuals. Longitudinal analyses showed that patients with primary BC who developed BM during follow-up had higher levels of TRACP5b (+95%, P=0.08) at the time of primary diagnosis, those patients had also a higher increases of ICTP (P=0.006), MMP-7 (P=0.004) and TIMP-1 (P=0.017) during follow-up than patients who did not progress toward bone metastasis. This study provides evidence of increase and interrelationship of circulating markers of angiogenesis, invasion and bone resorption in patients with BC with and without BM. Markers of bone resorption have the highest independent diagnostic value for detecting and potentially predicting BM in breast carcinoma patients.

Novel anticancer function of inositol hexaphosphate: inhibition of human rhabdomyosarcoma in vitro and in vivo.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that has been shown to suppress the growth of epithelial cancers, including those of breast and colon. The objective of this study was to investigate whether IP6 inhibits growth of rhabdomyosarcoma (RMS), a tumor of mesenchymal origin, which is the most common soft tissue sarcoma in children. We performed both in vitro and in vivo studies to evaluate the effect of IP6 on human RD cells growth. Our results show that IP6 suppresses growth of rhabdomyosarcoma cell line (RD) in vitro in a dose-dependent fashion. A 50% inhibition of cell growth (IC50) was induced by < 1.0 mM IP6. However, the removal of IP6 from the media, after 72 hours of treatment, allowed cells to recover their logarithmic growth. Exposure of RD cells to IP6 led to differentiation; cells became larger with abundant cytoplasm, expressing higher levels of muscle-specific actin. Consistent with in vitro observation, IP6 suppressed RD cell growth in vivo, in a xenografted nude mice model. When compared to controls, IP6-treated mice produced a 25 fold smaller tumors (p = 0.008), as observed after a two weeks treatment. In a second experiment, wherein the treatment period was extended to five weeks, a 49 fold (p = 0.001) reduction in tumor size was observed in mice treated with IP6. Histologically no evidence of tumor cell necrosis was observed. These data suggest a potential usefulness of this cytostatic, and non-cytotoxic, compound in novel therapeutic strategies for these types of tumor.

Expression of a kinase-deficient IGF-I-R suppresses tumorigenicity of rhabdomyosarcoma cells constitutively expressing a wild type IGF-I-R.

Previous results have shown that the insulin-like growth factor type I receptor (IGF-I-R) plays a critical role in the control of rhabdomyosarcoma (RMS) growth. The purpose of this study was to investigate whether a mutated IGF-I-R, when expressed in RMS cells, may interfere with the function of the endogenous wild-type IGF-I-R. We also examined whether the expression of a mutated IGF-I-R may induce phenotypic changes in RMS cells. We used here the mutated IGF-I-R with a lysine to arginine residue 1003 substitution, called IGF-I-KR, which carries a mutation in the ATP-binding domain of the intracellular beta subunit, while the extracellular, ligand binding alpha subunit remains unchanged. We observed that the expression of this mutated IGF-I-KR markedly decreased the response of RMS cells to stimulation with IGF-I. While stimulation with IGF-I increases the autophosphorylation of IGF-I-R in the parent cells, stimulation with IGF-I failed to produce a comparable increase in autophosphorylation in the cells expressing the mutated IGF-I-KR. We also observed a decreased plating efficiency of cells expressing the mutated IGF-I-KR. Consistently, a decrease of RMS growth in vivo was observed in an animal model. Our data suggest that the IGF/IGF-I-R signaling pathway may be inhibited by expressing a mutated IGF-I-KR and that such a mutant gene could be utilized in developing novel therapeutic strategies to suppress RMS growth. 1998.

Disruption of the MyoD/p21 Pathway in Rhabdomyosarcoma.

Purpose. Rhabdomyosarcoma (RMS) is an embryonal tumor thought to arise from skeletal muscle cells that fail to differentiate terminally. The majority of RMSs express MyoD, a protein essential to the differentiation of skeletal muscle. It was recently shown that during myogenesis, MyoD activates the expression of the cyclin-dependent kinase inhibitor (CDKi), p21, which itself plays a critical role in normal muscle development. To investigate the integrity of the MyoD/p21 pathway in RMS, we analyzed p21 and its relationship to MyoD expression in RMS.Methods. A panel of RMS samples was assembled from primary biopsies and from cell lines. Integrity of p21 was analyzed by single-strand conformation polymorphism (SSCP) and sequencing. Expression of p21 and MyoD was determined by Northern blot analysis, and the ability of exogenous p21 to arrest the cell cycle of RMS cell line was determined by transfection studies.Results. Our analysis indicates that although p21 is wild type in RMS, there is an inverse correlation between the levels of p21 and MyoD in these tumors. Tumors that express significant amounts of MyoD fail to express p21. This does not appear to be the result of mutations within the potential CACGTG sites present in the p21 promoter region or in the coding region of p21. An additional group of RMSs express very high levels of p21 but express little, if any, MyoD. Furthermore, RD, a RMS cell line which expresses high levels of endogenous p21, undergoes withdrawal from the cell cycle following forced expression of p21, suggesting that the pathway which would lead to G(1) arrest from endogenous p21 activity is defective.Discussion. These data suggest that the interaction between p21 and MyoD is defective in RMS although the precise nature of the defect remains to be elucidated.

The insulin-like growth factor-I receptor is required for EWS/FLI-1 transformation of fibroblasts.

Ewing's family of tumors is characterized by a well described reciprocal translocation, t(11;22)(q24;q12), which produces a fusion protein (EWS/FLI-1) that transforms mouse fibroblasts. The EWS/FLI-1 fusion protein has been shown to act as a potent chimeric transcription factor. Overexpression of insulin-like growth factor-I receptor (IGF-IR) has been implicated in many tumor models as playing a role in cell growth and tumorigenesis. In addition, blockade of the IGF-IR inhibits the growth of Ewing's family of tumors cells. Therefore, we first studied whether the presence of the IGF-IR is required for transformation by the EWS/FLI-1 fusion protein. To perform this study, we used two previously described fibroblast cell lines, R- and W, derived from an IGF-IR knockout mouse and a wild-type littermate, respectively. Neither W nor R- cells without the fusion protein formed soft agar colonies. However, W clones expressing the fusion message (WF cells) formed soft agar colonies, whereas R- clones expressing the fusion message (R-F cells) did not form soft agar colonies. Because the IGF-IR is required for EWS/FLI-1 transformation, we chose to investigate whether altered signaling occurs from the IGF-IR when the EWS/FLI-1 fusion is present. WF cells demonstrated a greater degree of ligand-stimulated insulin receptor substrate-1 phosphorylation when compared with W cells, suggesting that expression of the EWS/FLI-1 fusion protein alters the IGF-IR signaling pathway.

Suppression of rhabdomyosarcoma growth by fumagillin analog TNP-470.

The purpose of our study was to investigate a novel therapeutic approach for rhabdomyosarcoma (RMS) in an animal model. The pursuit of new therapeutic modalities for RMS is critically important since this type of tumor is the most common soft tissue sarcoma in children and because patients with metastatic disease may not be cured with current therapeutic modalities. We studied whether RMS growth may be suppressed by TNP-470, an analog of fumagillin, which was found to inhibit neoangiogenesis. Our data had shown that animals treated with TNP-470 (60 mg/kg), over a specific period of time, had approximately 50% smaller tumors than controls. Consistent with previous observations, treatment with TNP-470 decreases the level of the cyclin D1. Tumors dissected from TNP-470-treated animals had also considerable necrotic areas. In addition, TNP-470 had a direct cytotoxic effect on RMS cells in vitro. Our study has shown, therefore, that RMS in an animal model and in vitro responds to treatment with TNP-470, which suggests that the inhibitors of angiogenesis may be useful in a novel therapeutic design for RMS.

Human fetal glial cells constitutively produce HIV-inducing cytokines.

The capacity of two human fetal glial cell lines, SVG and POJ, to increase the expression of human immunodeficiency virus (HIV) was investigated. As a cellular model for HIV latency, a chronically infected promonocytic cell line U1 was used. This cell line constitutively expresses a low level of viral activity. To monitor the level of HIV expression in U1 cells, reverse transcriptase (RT) activity was measured in the supernatant and the level of total HIV proteins was determined in cellular lysates. It was observed that the conditioned media from SVG and POJ cells increased RT activity in U1 cells in a dose-dependent fashion. In addition, the conditioned media from fetal glial cells caused an increase in total HIV protein synthesis. The capacity of conditioned media from both fetal glial cell lines to induce the expression of HIV was reduced by 45% in the presence of antibodies against human tumor necrosis factor alpha (TNFalpha), suggesting that one of the HIV-activating factors released by these cells was TNFalpha. The presence of TNFalpha and two other HIV-activating cytokines, IL-6 and IL-1, was confirmed by ELISA. It was also observed that glutathione increased the HIV-inducing capacity of the fetal glial cell-derived conditioned media. The finding that fetal glial cells constitutively secrete soluble factors which increase the expression of HIV in vitro suggests that in vivo, during perinatally acquired infection, similar events may occur. Fetal glial cells may play an important role in the pathogenesis of HIV-related encephalopathy.

Tumorigenic and mitogenic capacities are reduced in transfected fibroblasts expressing mutant insulin-like growth factor (IGF)-I receptors. The role of tyrosine residues 1250, 1251, and 1316 in the carboxy-terminus of the IGF-I receptor.

Regulation of ligand-mediated signal transduction through transmembrane tyrosine kinase growth factor receptors involves phosphorylation of tyrosine residues in the intracellular domain of the receptor. The insulin-like growth factor-I (IGF-I) receptor contains three tyrosine residues in the carboxy-terminal domain at positions 1250, 1251, and 1316. Of these, only the tyrosine at position 1316 is conserved in the homologous position of the insulin receptor. Mutational analysis was used to study the role of these tyrosines in specific outcomes of IGF-I-mediated signal transduction. Mutations in the human IGF-I receptor were either replacement of tyrosines 1250 and 1251 with phenylalanine and histidine (yyFH), respectively, or replacement of the conserved distal tyrosine (position 1316) with phenylalanine (yCF). The yyFH mutation results in an IGF-I receptor with the amino acids found in the homologous position of the human insulin receptor. Cells overexpressing mutated IGF-I receptors were compared with cells expressing only endogenous IGF-I receptors or overexpressing wild-type IGF-I receptors. The ability of yyFH mutant IGF-I receptors to autophosphorylate the beta-subunit or phosphorylate insulin receptor substrate-1 was not significantly different from wild-type type IGF-I receptors. However, one or both of the proximal tyrosine residues (positions 1250 and 1251) in the carboxy-terminus of the IGF-I receptor are essential for IGF-I-stimulation of mitogenic and tumorigenic pathways. IGF-I-induced mitogenesis, measured as thymidine incorporation and cellular proliferation, was abrogated in cells overexpressing mutant IGF-I receptors with replacement of the proximal double tyrosines (positions 1250 and 1251). Fibroblasts expressing this mutant IGF-I receptor formed fewer tumors than the negative control cells, whereas cells expressing wild-type IGF-I receptors formed large tumors in all recipient mice injected. Conversely, cells expressing mutant IGF-I receptors with only the conserved distal tyrosine (position 1316) replaced had slightly reduced IGF-I-stimulated beta-subunit autophosphorylation, thymidine incorporation, and cellular proliferation when compared with cells expressing wild-type receptors. Phosphorylation of insulin receptor substrate-1 by the yCF mutant receptors was not impaired. Despite the ability of these mutant receptors to stimulate mitogenic growth, fibroblasts expressing this mutant receptor were also incapable of forming tumors in recipient nude mice. The distal tyrosine (position 1316) of the IGF-I receptor is crucial for tumor formation but is not essential for IGF-I stimulated mitogenesis. Thus, the tyrosine moieties in the carboxy-terminus of the IGF-I receptor participate in the signal transduction pathways that affect the mitogenic and tumorigenic potentials of cells expressing mutant IGF-I receptors.

1 alpha, 25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531) modulation of insulin-like growth factor-binding protein-3 and induction of differentiation and growth arrest in a human osteosarcoma cell line.

1 alpha,25-Dihydroxycholecalciferol [1,25-(OH)2D3] is a potent differentiating agent in a variety of tumor cell lines. However, the induction of severe hypercalcemia has limited its clinical use. Several analogs have been synthesized that retain the antiproliferative differentiating effects of 1,25-(OH)2D3, but do not have the calcitropic effect of the parent compound. One such analog, 1 alpha,25(OH)2-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531), can induce differentiation in HL-60 cells and does not induce hypercalcemia in animal models. We, therefore, evaluated the effect of Ro24-5531 on a human osteosarcoma cell line, MG-63. Compared with 1,25-(OH)2D3, the analog Ro24-5531 is 10-100 times more potent as an inhibitor of MG-63 cell proliferation, as determined by [3H]thymidine incorporation and/or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The inhibition in cell growth is accompanied by a decrease in the expression of p34cdc2 (> 4-fold), a protein critically involved in cell cycle regulation. Ro24-5531 treatment of MG-63, at a concentration of 10(-8) mol/L, induced expression of the bone differentiation markers biglycan and osteocalcin, as determined by Northern analysis. These data suggest that Ro24-5531 treatment induces growth arrest coupled with differentiation. To begin to evaluate the mechanisms by which Ro24-5531 may exert an effect, we evaluated the effect of Ro24-5531 on components of the insulin-like growth factor I (IGF-I) signaling pathway, an important regulator of normal bone growth and differentiation. The expression of IGF-binding protein (IGFBP), IGFBP-3 messenger ribonucleic acid, and protein levels are increased 20-fold after 72 h of treatment with Ro24-5531 and are associated with a marked increase in detectable binding of ligand to binding protein, as measured by RRA. These data suggest an association between Ro24-5531-induced growth arrest and increased expression of IGFBP-3.

Metastatic human rhabdomyosarcoma: molecular, cellular and cytogenetic analysis of a novel cellular model.

A new human metastatic rhabdomyosarcoma (RMS) model was established and analyzed for a number of biologic, cytogenetic and molecular parameters. Consistent with previous studies, the metastatic capacity of different RMS cell variants did not correlate with their tumorigenic or proliferative capacities. Interestingly, a highly metastatic variant was diploid, while a nonmetastatic variant was tetraploid, which parallels previous clinical observations. Genes whose expression had been found to be associated with either low- or high-metastatic capacity in carcinoma or melanoma did not show a similar association with different metastatic variants of RMS, derived from a mesenchymal tumor. We also found, in transient reporter gene assays, that several promoters had higher transcriptional activity in highly metastatic than in nonmetastatic RMS cell variants. This novel human RMS metastatic model may be instrumental for a better understanding of the regulatory pathways that control the metastatic phenotype of tumors of mesenchymal origin.

The role of the tyrosine kinase domain of the insulin-like growth factor-I receptor in intracellular signaling, cellular proliferation, and tumorigenesis.

Insulin and insulin-like growth factor (IGF-I) receptors are heterotetrameric proteins consisting of two alpha-and two beta-subunits and members of the transmembrane tyrosine kinase receptors. Specific ligand binding to the receptor triggers a cascade of intracellular events, which begins with autophosphorylation of several tyrosine residues of the beta-subunit of the receptor. The triple cluster in the tyrosine kinase domain of the beta-subunit is the earliest and major autophosphorylation site. Previous studies have shown that substitutions of these three tyrosines by phenylalanines of both insulin and IGF-I receptors practically abolish any activation of cellular signaling pathways. We have studied the effect of double tyrosine mutations on IGF-I induced receptor autophosphorylation, activation of Shc and IRS-1 pathways, and cell proliferation and tumorigenicity. Substitution of tyrosines 1131/1135 blocks any detectable autophosphorylation, whereas substitution of tyrosines 1131/1136 or 1135/1136 only reduces autophosphorylation levels in some clones by approximately 50%. Nevertheless, all the cells expressing IGF-I receptors with double tyrosine substitutions demonstrated markedly reduced signaling through Shc and IRS-1 pathways. In addition, they were unable to respond to IGF-I-stimulated cell growth in culture, and tumor formation in nude mice was abrogated. These data suggest that the presence of tyrosine 1131 or 1135 essential for receptor autophosphorylation, whereas the presence of each of these tyrosines is necessary for a fully functional receptor.

HIV-1 modulates the expression of gelatinase A and B in monocytic cells.

The levels of mRNA of both gelatinases A and B were dramatically decreased in HIV-infected cells, when compared to uninfected cells. The expression of gelatinase A in HIV-infected cells was selectively increased by tumor necrosis factor (TNF alpha) while the expression of gelatinase B was not affected. In contrast, in uninfected cells TNF alpha down regulated gelatinase B mRNA level, without affecting the gelatinase A. N-acethylcysteine (NAC) increased the levels of mRNA of both gelatinases. The conditioned media from HIV-infected and uninfected cells had comparable level of secreted gelatinase A protein. These data suggest that in monocytic cells different regulatory pathways control gelatinases A and B and that HIV could modulate in vivo the expression of these proteolytic enzymes, critically involved in regulation of invasion of basement membrane.

In vivo treatment with antibody against IGF-1 receptor suppresses growth of human rhabdomyosarcoma and down-regulates p34cdc2.

In a previous study, we have shown that insulin-like growth factor type 2 (IGF-2) functions as an autocrine growth factor in human rhabdomyosarcoma (RMS) cell lines. In addition, we demonstrated that the inhibition of binding of IGF-2 to the IGF-1 receptor, mediated by suramin, blocked the growth of RMS cells in vitro. We now report that, in vivo, a specific IGF-1 receptor blocking antibody (alpha IR-3), but not suramin, suppresses RMS tumor growth. Both progression of tumor growth in tumor-bearing animals and formation of newly established tumors were suppressed by treatment with alpha IR-3. Histological analysis of tumors from treated animals did not reveal necrotic lesions, implying that the treatments had no cytotoxic effect. The decrease in tumor growth was associated with a decrease of p34cdc2, a cellular protein involved in cell cycle regulation, suggesting that treatment resulted in the arrest of cellular proliferation. We speculate, therefore, that agents which block the IGF signaling pathway may find application in treatment of RMS.

Organic thiophosphate WR-151327 suppresses expression of HIV in chronically infected cells.

Reducing agents such as glutathione (GSH), glutathione ester (GSE), and N-acetylcysteine (NAC) have been shown to suppress the induction of HIV expression in chronically infected cells stimulated by cytokines. We present data which show the effects of the organic thiophosphate WR-151327 on the expression of latent HIV in U1 cells. The chronically infected promonocytic cell line U1 constitutively expresses low levels of HIV that can be increased by 13-phorbol 12-myristate acetate (PMA), tumor necrosis factor alpha (TNF-alpha), and granulocyte/monocyte colony-stimulating factor (GM-CSF). WR-151327 suppressed, in dose-dependent fashion, the reverse transcriptase (RT) activity induced by TNF-alpha, GM-CSF, and PMA. The maximal decrease in RT activity was 70, 80, and 50%, respectively. Pretreatment with WR-151327 also suppressed the induction of total HIV protein synthesis, as shown by Western blot analysis. In addition, WR-151327 suppressed HIV-LTR-CAT activity in transfected human rhabdomyosarcoma cells (RD). Suppression of HIV expression by WR-151327 was observed in the absence of a cytotoxic or cytostatic effect. Incubation of WR-151327 with human recombinant TNF-alpha for 6 hr at 37 degrees C did not alter the capacity of TNF-alpha to induce the expression of HIV. Our observations further support the hypothesis that reducing agents are important in the control of HIV replication and that the clinical evaluation of WR-151327 may be indicated.

Ara-C treatment leads to differentiation and reverses the transformed phenotype in a human rhabdomyosarcoma cell line.

Rhabdomyosarcoma (RMS) is an embryonal tumor of childhood that arises from primitive skeletal muscle-forming cells (rhabdomyoblasts) probably arrested and transformed along the normal myogenic pathway to maturation. Since Ara-C is an antitumor agent known to induce differentiation in human acute myelogenous leukemia, also presumably a disorder of cellular maturation, we treated RD, a human embryonal RMS cell line, with Ara-C and evaluated its effect on growth and differentiation. Ara-C treatment of RD cells in vitro caused a dose-dependent growth inhibition in the absence of cytotoxicity. Interestingly, RD cells treated with 5 x 10(-7) M Ara-C for 4 days were able to recover logarithmic growth after the removal of the drug from the media. A reexposure of these cells to Ara-C led to morphological and biochemical changes related to differentiation, including the appearance of an increased number of multinucleated cells that expressed muscle-specific actin and skeletal muscle myosin heavy chain (MHC) (fast). In vivo studies demonstrated that RD cells pretreated with 5 x 10(-7) M Ara-C lost their ability to form tumors in nude mice. We conclude that treatment of this human embryonal RMS cell line with Ara-C results in marked growth inhibition in vitro, loss of tumorigenicity in vivo, and the expression of biochemical markers present in a more differentiated phenotype. These data suggest a potential role for differentiation therapy as a therapeutic approach in RMS.

Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine.

The effects of glutathione (GSH), glutathione ester (GSE), and N-acetyl-L-cysteine (NAC) on the induction of human immunodeficiency virus (HIV) expression were investigated in the chronically infected monocytic U1 cell line, a previously described cellular model for HIV latency. U1 cells constitutively express low levels of virus, which can be increased by phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and other inducers. GSH, GSE, and NAC suppressed in a dose-dependent fashion the induction of HIV expression mediated by PMA, TNF-alpha, and IL-6, in the absence of cytotoxic or cytostatic effects. Reverse transcriptase activity, inducible by PMA, TNF-alpha, or IL-6, was decreased by 80-90% after pretreatment with GSH, GSE, or NAC. The induction of total HIV protein synthesis was also decreased appreciably after pretreatment with GSH, GSE, or NAC. The accumulation of HIV mRNA was substantially suppressed after pretreatment with NAC but to a lesser extent after pretreatment with GSH or GSE. Although PMA induces the expression of TNF-alpha in U1 cells, the suppressive effect of GSH, GSE, and NAC on PMA-induced HIV expression in U1 cells was not associated with the inhibition of TNF-alpha expression. The present findings, which elucidate relationships between cellular GSH and HIV expression, suggest that therapy with thiols may be of value in the treatment of HIV infection.

Astrocyte-conditioned medium stimulates HIV-1 expression in a chronically infected promonocyte clone.

Human promonocytic cells chronically infected with human immunodeficiency virus-1 (HIV-1) (clone U1.1.5) were grown in the presence of media conditioned by primary rat cortical astrocytes and HIV-1 expression was assessed by measuring reverse transcriptase activity. Media conditioned by non-stimulated and lipopolysaccharide (LPS)-stimulated astrocytes induced the expression of HIV-1 2.1-fold and 4.1-fold, respectively. LPS alone, media conditioned by the uninfected parental cell line of U1.1.5 (U937), and culture media from four other cell lines, had no effect on viral expression. The magnitude of induction was time- and dose-dependent. Tumor necrosis factor alpha (TNF-alpha) was detected in LPS-stimulated astrocyte-conditioned medium and the HIV-inducing capability of the medium was neutralized, in part, by an antibody to recombinant murine TNF-alpha. These results suggest a role for astrocytes in the induction of HIV expression and thus in the pathogenesis of HIV-1 infection in brain.